[LCN2 knockout protects endothelial barrier function in mice with sepsis-induced acute lung injury by regulating the NF-κB signaling pathway].

摘要

OBJECTIVES: To investigate the protective effect of lipocalin-2 (LCN2) gene knockout against sepsis-induced acute lung injury (ALI) and vascular endothelial dysfunction in mice and the mediating role of the nuclear factor-κB (NF-κB) signaling pathway. METHODS: Wild-type (WT) C57BL/6 mice receiving sham operation or cecal ligation and puncture (CLP) to induce sepsis were randomized into 3 subgroups for intraperitoneal injections of saline, PDTC, or Bay 11-7082 (n=10). Twenty LCN2 knockout (LCN2KO) mice were randomized for sham operation or CLP modeling. Pulmonary histopathological changes, lung wet-to-dry (W/D) ratio, lung index, Evans blue extravasation, and serum TNF-α and IL-6 levels in the mice were assessed. The colocalization of NF-κB, ICAM-1, and VCAM-1 with CD31 were analyzed using immunofluorescence staining, and endothelial cell apoptosis was examined with TUNEL/CD31 double staining. Western blotting and co-immunoprecipitation assay were performed to analyze the protein expressions of LCN2, p-P65/P65, ICAM-1, VCAM-1 and VE-cadherin and the interaction between LCN2 and P65. RESULTS: The WT mice receiving CLP showed severe lung structural damage with obvious edema and inflammation. In contrast, both the LCN2KO mouse and PDTC-treated WT mouse models of CLP showed milder lung injury with lower W/D ratio, lung index, Evans blue leakage, serum TNF‑α and IL-6 levels and markedly reduced colocalization signals of NF-κB, ICAM-1, and VCAM-1 with CD31. CLP resulted in significantly increased expression levels of LCN2, p-P65/P65, ICAM-1, and VCAM-1, lowered VE-cadherin levels, and enhanced endothelial cell apoptosis, and all these changes were significantly ameliorated in LCN2KO mice and WT mice with NF‑κB inhibitor treatment. Co-immunoprecipitation results suggested the interaction between lung LCN2 and P65. Bay 11-7082 significantly reduced inflammatory cytokine production, downregulated p-P65 expression and its downstream adhesion molecules, and restored VE-cadherin expression. CONCLUSIONS: LCN2 knockout alleviates sepsis-induced ALI and vascular endothelial dysfunction in mice by inhibiting NF-κB signaling. 目的: 探讨敲除脂质运载蛋白2（LCN2）基因对脓毒症小鼠急性肺损伤及血管内皮功能障碍的影响，明确核因子-κB（NF-κB）通路在其中的可能作用。方法: 选用20只LCN2基因敲除（LCN2KO）小鼠和60只C57BL/6小鼠，通过盲肠结扎与穿孔（CLP）术构建脓毒症模型，分为WT-Sham组、Sham+PDTC组、Sham+Bay 11-7082组、WT-CLP组、LCN2KO-Sham组，LCN2KO-CLP组、CLP+NF-κB抑制剂PDTC组、CLP+NF-κB抑制剂Bay 11-7082组，10只/组。HE染色观察肺组织病理变化，检测肺湿干比（W/D）、肺指数及伊文思蓝外渗评价肺水肿与血管通透性。ELISA测定血清肿瘤坏死因子-α（TNF-α）及白细胞介素-6（IL-6）水平。免疫荧光检测NF-κB、细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）与CD31共定位情况，TUNEL/CD31双染评估内皮细胞凋亡变化。Western blotting检测LCN2、p-P65/P65、ICAM-1、VCAM-1及血管内皮钙黏附蛋白（VE-cadherin）蛋白表达。免疫共沉淀分析LCN2与P65的潜在相互作用。结果: WT-CLP组小鼠肺组织结构明显受损，伴随水肿及炎症反应。与WT-CLP组相比，LCN2KO-CLP组及CLP+PDTC组小鼠肺组织损伤减轻，W/D值、肺指数及伊文思蓝外渗量下降，血清TNF-α和IL-6水平降低，肺血管内皮细胞中NF-κB、ICAM-1、VCAM-1与CD31的共定位信号减弱。CLP术后LCN2、p-P65/P65、ICAM-1及VCAM-1表达升高，VE-cadherin表达降低，内皮细胞凋亡增加，LCN2KO及抑制剂干预后相关蛋白表达得到改善，凋亡水平下降。免疫共沉淀结果显示LCN2与P65在肺组织中可能存在相互作用。Bay 11-7082干预后，降低了炎症因子，下调了p-P65及其下游黏附分子表达，恢复VE-cadherin水平。结论: 敲除LCN2可通过抑制NF-κB信号通路减轻脓毒症诱导的急性肺损伤及血管内皮功能障碍。.